1. Field of the Invention
The present invention relates to an image detecting method. For example, the present invention relates to a method and an apparatus which capture fluorescence-labeled oligonucleotide in plural positions on a plane, and which then detect a fluorescence intensity and a fluorescence pattern on the basis of the captured oligonucleotide.
2. Description of the Related Art
The techniques of analyzing DNA, RNA, protein and so on are important in the fields of medicine, biology and the like, including gene analysis and gene diagnosis. Particularly in recent years, attention has been given to a method and an apparatus for simultaneously examining and analyzing various types of DNA sequence information and genetic information from one analyte, by using a DNA microarray (also defined as various names, such as an oligochip, a DNA chip and a biochip, which will be hereinafter collectively called a “DNA microarray”). The DNA microarray is formed by using a glass board or the like, which is divided into plural regions (e.g., several hundreds of regions to several tens of millions of regions), each of which is provided with a target DNA probe (generally, the DNA probes are of different types) immobilized thereon to form a minute reaction region. When the reaction region is caused to react with an analyte, an object DNA in the analyte is hybridized with, and captured by the immobilized DNA probe. When a fluorescence probe or the like is further bonded to the target DNA, fluorescence intensity or the like can be measured to determine a bonded state (i.e., a bonded position, which means a hybridized sequence) and the amount of bonding. The measurement results can be utilized for gene diagnosis, sequencing and so on.
An apparatus similar to a microscope (e.g., a confocal fluorescence microscope) generally called a “scanner” is used for reading the fluorescence intensity of the DNA microarray (e.g., JP-A No. Hei 9-503308 and JP-A No. 2000-69998). This apparatus irradiates the array with a minute spot beam of excitation light, such as laser light, then separates emitted fluorescence from the excitation light by using a spectroscope such as an interference filter, and then detects the fluorescence intensity by means of a photodetector such as a photomultiplier tube. In this event, the apparatus can determine the distribution of fluorescence intensity throughout the array, that is, the degree of bonding to each DNA probe, by two-dimensionally scanning the relevant minute spot formed on the array by use of a galvanometer mirror or the like, or by two-dimensionally scanning the array with the position of the minute spot fixed. Methods of reading the fluorescence intensity of the DNA microarray include, in addition to the above beam scanning, the method for irradiating the regions of the array with excitation light over a wide range, and then detecting a emitted fluorescent image by means of a two-dimensional camera (e.g., JP-A No. 2002-181708 and JP-A No. 2001-255328). The each region having the DNA probe immobilized is divided into several tens of sub regions, and the fluorescent intensity of the sub regions are detected. And regions except DNA probe immobilized regions are divided into sub regions.
In these methods, the detection of the fluorescence on the plural regions each having the DNA probe immobilized thereon involves making measurements on the array as divided into several tens of regions, and making measurements on each of the regions as subdivided. This makes it possible to detect the fluorescence on the regions as isolated from one another, even in a case where the plural reaction regions are misaligned. However, the number of pixels required for the two-dimensional sensor is several hundreds or more times the number of regions to be measured.
Determining of DNA, RNA sequencing is also an important technique. Samples are generally prepared in advance by labeling, with fluorescence, DNA fragments or groups of DNA fragments used in sequencing, and a molecular weight pattern in separation and expansion is measured and analyzed after electrophoretic migration or in electrophoretic migration. Specifically, a well-known Sanger method is used for inducing a dideoxy reaction, prior to the electrophoresis. Oligonucleotide with about 20 bases long, complementary to a known portion of a base sequence of sample DNA to be analyzed, is synthesized and labeled with a fluorophore. This oligonucleotide is used as a primer to form a complementary chain bond with about 1 picomole of the sample DNA, and thus yielding polymerase, which in turn induces a complementary chain extension reaction. Here, four types of deoxynucleotide triphosphates, namely, dATP, dCTP, dGTP and dTTP, as well as ddATP, for example, are added as substrates. When ddATP is captured with the complementary chain extension, no further extension of the complementary chain takes place. Thus, the fragments of varying lengths terminating with adenine (A) are prepared. For the above reaction, ddCTP, ddGTP and ddTTP are added in place of ddATP to induce reactions. The primers used in the each reactions have the same base sequence, are labeled with four types of fluorophores each of which can be spectroscopically identified. When the above four types of reactants are mixed, fragments with up to several hundreds of bases long and of lengths varying base by base, which fragments are complementary to the sample DNA, are obtained as labeled with the four types of fluorophores varying depending on the type of terminal base. The fragments are separated with a resolving power of one base, by capillary gel electrophoresis. The obtained samples migrate while being separated, and are irradiated with laser in order from the shortest sample. When fluorescence emissions are spectroscopically measured by using plural filters, the types of terminal bases of all fragments can be determined in order from the shortest fragment, on the basis of the temporal change in the fluorescence intensities of the respective four types of fluorescent substances.
Recently proposed is an approach of fixing DNA or the like to a board for sequencing, as described in Proc. Natl. Acad. Sci. (Proceedings of National Academy of Sciences), USA, vol. 100 (7), pp. 3960, 2003. With this approach, sequencing is performed by randomly capturing, molecule by molecule, fragments of sample DNA to be analyzed on a surface of the board, then inducing extension for substantially every base, and then detecting results thereof by fluorescence measurement. Specifically, sequencing of the sample DNA is performed by repeating a cycle including the steps of: inducing a DNA polymerase reaction, by using four types of dNTP derivatives (MdNTP) with detectable labels, which are captured as substrates of DNA polymerase in template DNA, and which can terminate a DNA chain extension reaction with the presence of a protecting group; then detecting the captured MdNTP on the basis of fluorescence or the like; and then returning the MdNTP to an extension-capable state. Since this technique allows sequencing of DNA fragments molecule by molecule, the technique makes it possible to concurrently analyze many fragments. Hence, analysis throughput can be increased. Since this method may possibly make it possible to perform sequencing by DNA single molecule, the method may possibly eliminate a problem inherent in the prior art, that is, the need to refine or amplify sample DNA for cloning, PCR or the like. Thus, speedy genome analysis and gene diagnosis can be expected. In the method, the molecules of the fragments of the sample DNA to be analyzed are randomly fixed on the surface of the board. For this reason, the method requires an expensive camera having the number of pixels that is several hundreds of times the number of the trapped molecules of the DNA fragments. Specifically, when the molecules of the DNA fragments are adjusted as spaced apart from one another at average intervals of 1 micron, some molecules are spaced apart from one another at greater intervals, and others are spaced apart at smaller intervals. Consequently, in order to detect the molecules as isolated from one another, fluorescent intensities need to be detected at a minute interval (example, <0.1 μm). Generally, resolution of measuring fluorescent image must be several tenth of the interval between the DNA molecules.